College of Life Science and Bioengineering, Beijing University of Technology, No.100, PingLeYuan, ChaoYang District, Beijing, , P.R.
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1 Supporting information A T 1 -Mediated Nanosensor for Immunoassay Based on Activatable MnO 2 Nano-Assembly Zixin Liu, #, Yunlei Xianyu #, Wenshu Zheng #, Jiangjiang Zhang, Yunjing Luo *, Yiping Chen *, Mingling Dong, Jing Wu, and Xingyu Jiang, * College of Life Science and Bioengineering, Beijing University of Technology, No.100, PingLeYuan, ChaoYang District, Beijing, , P.R. China Beijing Engineering Research Center for BioNanotechnology & CAS Key Laboratory for Biological Effects of Nanomaterials and Nano-safety, CAS Center for Excellence in Nanoscience. National Center for NanoScience and Technology. 11 BeiYiTiao, ZhongGuanCun, Beijing , P.R. China The University of Chinese Academy of Sciences, 19 A YuQuan Road, ShiJingShan District, Beijing, , P. R. China * Corresponding author. Tel: addresses: luoyj@bjut.edu.cn (YJ Luo), chenyp@nanoctr.cn (YP Chen), and xingyujiang@nanoctr.cn (XY Jiang) # These authors contribute to this work equally. S-1
2 Content list Experimental section Table S Fig.S Fig.S Fig.S Fig.S S-2
3 1. Materials and Equipment 1.1 Materials Manganese acetate tetra-hydrate (MnAC 2, MW=245.09), N-hydroxysulfosuccinimide sodium salt (NHS), sodium L-ascorbate (MW=198.11), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and bovine serum albumin (BSA) are from Aladdin Co., Ltd. (Shanghai, China). Sodium bismuth oxide (NaBiO 3, MW=279.97) is from Alfa Aesar Co., Ltd. (Shanghai, China). TCO-PEG 4 -NHS ester (TCO) and tetrazine-sulfo-nhs ester (Tz) are from Click Chemistry Tools LLC. (USA). Carboxyl magnetic beads (MBs) (1000 nm, MBs content: 10 mg/ml) are from Thermo Fisher Scientific Co., Ltd. (Shanghai, China). Carboxyl magnetic iron oxide nanoparticles (MNPs, 30nm, solid content: 5 mg/ml) and conjugation kits are from Ocean Nano Tech LLC. (USA). MES monohydrate free acid (MES, MW=213.2) is from MP Biomedicals LLC. (USA). NaOH and N, N-dimethylformamide (DMF) are from Beijing Chemical Reagents Co., Ltd. (Beijing, China). Tris-HCl (MW=157.64) is from LanYi Reagent Co., Ltd. (Beijing, China).We purchase 3, 3, 5, 5 -tetramethylbenzidine (TMB) from Beyotime Co., Ltd. (Shanghai, China). We purchase the procalcitonin (PCT, 1.4 mg/ml), PCT capture antibody (Ab 1, 5 mg/ml), PCT detection antibody (Ab 2, 5 mg/ml), interleukin-6 (IL-6, 2.4 mg/ml), C-reactive protein (CRP, 5 mg/ml) and human IgG (IgG, 10 mg/ml) from Sino Biological Inc. (Beijing, China). The phosphates tablets and Tween-20 are from Amresco LLC. (USA). The clear-bottom 96-well microplates are from Corning Inc. (USA). We purchase the ultra-filtration unit (10 kd filter and 100 kd filter) from S-3
4 Millipore Co. (USA). Ultra-pure water (18.2 MΩ cm 1 ) used in our research is from the Millipore purification system (USA). All experiments were performed in compliance with the relevant laws and institutional guidelines. 1.2 Equipment A 1.5T cute nuclear magnetic resonance (NMR) analyzer for measurement of longitudinal relaxation time (T 1 ) and transverse relaxation time (T 2 ) is from Shanghai Shinning Globe Science and Education Equipment Co., Ltd. (Shanghai, China). Matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF-MS) for characterization of molecular weight is from Bruker Daltonics Inc. (USA). We employ Zetasizer Nano ZS (Malvern Instruments Co., Ltd.) to analyze the size of BSA-MnO 2 NPs and MnO 2 nano-assembly. We use a field emission transmission electron microscope (JEM-2100F, Japan) and a high resolution transmission electron microscope (Tecnai G 2 F20 S-TWIN, USA) to characterize the BSA-MnO 2 NPs. We use a multi-mode microplate reader (BioTek instruments Inc.) to investigate the enzyme-mimicking activity of BSA-MnO 2 NPs. We perform the X-Ray photoelectron spectroscopy (XPS) analyses on a Thermo Scientific ESCALAB 250XI photoelectron spectrometer with a monoachromatized X-ray source (powered at 20 ma and 10 KV). S-4
5 2. Supplementary Results Table S1. The results of MnO 2 -T 1 sensor, Roche-ECL method and T 2 -MRS sensor for detection of PCT in real serum samples. Sample No. MnO 2 -T 1 sensor (ng/ml) Roche-ECL method (ng/ml) T 2 -MRS sensor (ng/ml) 1 0.2± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±2.80 S-5
6 Figure S1. The change of T 1 value upon the addition of HCl into BSA-MnO 2 solution. S-6
7 Figure S2. The effect of ascorbic acid (AA) to magnetic beads. 0.6 mg/ml (100 µl) of magnetic beads and 0.5 mm (100 µl) of AA are mixed together for different time points (0, 10, 20, 30, 40, 50 and 60 min). The T 1 value of supernatant is measured after the magnetic separation. S-7
8 Figure S3. Characterization of the enzyme-like activity of BSA-MnO 2 NPs. (A) The time-dependent absorbance change at 652 nm of TMB with BSA-MnO 2 NPs or BSA. (B) Photograph of TMB in BSA-MnO 2 NPs solution, BSA solution and H 2 O. We take this photo using the a digital camera (Nikon D90, Nikon Corporation, Tokyo, Japan) S-8
9 Figure S4. Optimization of conditions of MnO 2 -T 1 sensor for detection of PCT. (A) and (B) The concentration of BSA-MnO 2 -Tz and BSA-MnO 2 -TCO; (C) Amount of Ab 2 -Tz; (D) The concentration of MBs-Ab 1 conjugate; (E) The concentration of AA; (F) The reaction time. S-9
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